Wastewater Surveillance to Confirm Differences in Influenza A Infection between Michigan, USA, and Ontario, Canada, September 2022–March 2023

Wastewater surveillance is an effective way to track the prevalence of infectious agents within a community and, potentially, the spread of pathogens between jurisdictions. We conducted a retrospective wastewater surveillance study of the 2022–23 influenza season in 2 communities, Detroit, Michigan, USA, and Windsor-Essex, Ontario, Canada, that form North America’s largest cross-border conurbation. We observed a positive relationship between influenza-related hospitalizations and the influenza A virus (IAV) wastewater signal in Windsor-Essex (ρ = 0.785; p<0.001) and an association between influenza-related hospitalizations in Michigan and the IAV wastewater signal for Detroit (ρ = 0.769; p<0.001). Time-lagged cross correlation and qualitative examination of wastewater signal in the monitored sewersheds showed the peak of the IAV season in Detroit was delayed behind Windsor-Essex by 3 weeks. Wastewater surveillance for IAV reflects regional differences in infection dynamics which may be influenced by many factors, including the timing of vaccine administration between jurisdictions.


Sample Processing
Composite samples of raw wastewater were concentrated by filtering 50-120 mL through 0.22 μm Sterivex PES cartridge filters (MilliporeSigma, Burlington, MA, USA) using a 60 mL syringe fitted into a caulking gun.Immediately following filtration, the filters were sealed and flash-frozen through immersion in liquid nitrogen.Subsequently, filters were subjected to downstream processes including RNA extraction and RT-qPCR.
Following filtration and flash freezing, filters were thawed, and the filter membrane was cut from the Sterivex cartridge using a sterile scalpel and forceps.Total nucleic acid was extracted from the filter membranes using either the AllPrep PowerViral DNA/RNA kit (Qiagen, Germantown, MD, USA) modified by addition of 5% 2-mercaptoethanol (v/v) or the RNeasy PowerMicrobiome Kit (Qiagen, Germantown, MD, USA), again modified by addition of 5% 2mercaptoethanol (v/v).Samples were not treated with DNase upon extraction and RNA was eluted in 50μL of RNase free water.

RT-qPCR
An RT-qPCR assay was used to measure the concentration of influenza A virus (IAV) in wastewater samples.The assay targeted RNA that codes for the matrix protein 1 (M1-gene) of IAV using primers and probes developed by the U.S. CDC (1).Primers and probes were supplied by Integrated DNA Technologies (Coralville, IA, USA) and primer and probe sequences can be found in Supplementary Table 1.

Amplicon Sequencing
To validate the identity of the amplicon obtained from M1 gene of IAV, RTq-PCR products obtained from IAV-positive wastewater samples were sequenced.First, the PCR products were cleaned up by adding 1 volume of NEBNext ® Sample Purification Beads (NEB). Spectrophotometer.
Second, the end-prep reaction was performed employing 250-300 ng of DNA mixed with (min_hit_len) and the minimum summed length of partial hits is set to 0 (6).Manual calculation of the identity % for the taxonomical assignments was not completed.The Centrifuge classification results are then filtered and aggregated to calculate and report counts of reads at the species rank.For reads without a reliable assignment at the species rank, higher ranks of the

1 .
75 μL UltraII End Prep Buffer, 0.75 μL UltraII End Prep Enzyme and water to a final volume of 15 μL.The reaction was incubated at 20°C for 10 minutes and 65°C for 10 minutes, holding at 4°C.Barcoding of samples was carried out combining 3 μL of end-prepped sample, 2.5 μL Native Barcode (ONT, native barcoding EXP-NBD104), 10 μL Blunt/TA Ligase Master Mix and 4.5 μL nuclease-free water.Samples were pooled after barcoding and then analyzed in a single sequencing run with separated reads obtained from each of the amplicons.Ligation reaction was incubated at 22°C for 20 minutes and 65°C for 10 minutes, followed by a hold on ice for at least 1 minute.Cleaning up of the ligated sample was performed by adding 0.4 volume NEBNext ® Sample Purification Beads and eluting with 12 μL of nuclease-free water.The Oxford Nanopore sequencing adaptor ligation was performed employing 200 ng of barcoded DNA in 30 μL, mixed with 5 μL Adaptor Mix II, 10 μL 5X NEBNext Quick Ligation Reaction Buffer (NEB) and 5 μL Quick T4 DNA Ligase (NEB).The incubation was carried out at 25°C for 30 minutes.Sample was cleaned up by adding 1 volume of NEBNext ® Sample Purification Beads, eluted in 12 μL of Elution Buffer and quantified.Twenty ng of the library was loaded onto a SpotOn Flow Cell (R9.4 flow cell).Data was collected along 16 hours of sequencing withMinION.The FastQ files containing the sequenced reads were uploaded in Epi2me desktop application (Oxford Nanopore Technologies).Reads were analyzed employing the WIMP (What's In My Pot) workflow (v2023.06.13-1865548) setting filters for reads length between 100 bp to 250 bp.WIMP initially filters FASTQ files with a mean q-score below a minimum threshold (defaults to 7).For reads above the quality threshold, the Centrifuge classification engine is executed to assign each read to a taxon in the NCBI taxonomy.Taxonomical assignment is done based on the scores calculated by the microbial classification engine called Centrifuge using default settings where the minimum length of partial hits is set to 25

Table 1 .
(5)(3)(4), corresponding to a greater than 95% probability of detection.LOD was determined through analysis of 20 replicate 8-point standard curves.Twist Synthetic Influenza H3N2 RNA control (Twist Bioscience, San Francisco, CA) was used to create an 8-point standard curve to quantify gene targets.RT-qPCR assays were also performed to evaluate the levels of Pepper Mild Mottled Virus (PMMoV) within the wastewater.PMMoV is a widely accepted indicator of the presence of human fecal matter(2)(3)(4).For quantification of PMMoV, reactions contained 2.5μL of RNA template mixed with 10μL of Luna Universal Probe One-Step Reaction Mix (2X), 1μL Luna WarmStart ® RT Enzyme Mix (20X) (Luna ® One-Step RT-qPCR Kit, Massachusetts, USA), 3.5μL of water and the remaining 3µL consisted of forward primer, reverse primer, and probe each with a final concentration of 200nM.Primers and probes for the amplification of PMMoV were previously described(5).Reverse transcription was performed for 10 minutes at 55°C, this was followed by an enzyme activation step at 95°C for 1 minute and 40 cycles of denaturation and annealing/extension at 95°C for 10 seconds and 55°C for 30 sec, then 55°C for 45 sec, respectively.No template controls yielded no amplification, and the limit of detection for the assay was determined at 4 gene copies of IAV per reaction containing 4μL of template seconds respectively.No template controls were included with each plate of RT-qPCR run and whole process controls were included with each extraction.The 7-point standard curve for the quantification of PMMoV was generated through serial dilution of a custom gBlock (Integrated DNA Technologies, Coralville, IA, USA) and was run with each plate of samples.No amplification was observed either process controls (extraction blanks) or in no template controls.Reaction inhibition was assessed using VetMAX XENO Internal Positive Control RNA (Applied Biosystems Corp., Waltham, MA, USA).VetMax template was spiked into water (which was used as a reference), undiluted DNA/RNA extracts, DNA/RNA extracts diluted 1:5, and DNA/RNA extracts diluted 1:10.Recovery was compared between conditions, and it was determined that inhibition could be addressed through dilution.Due to repeated incidence of inhibition with wastewater samples processed by filtration, template was diluted 1:5 or 1:10 in all reactions.Technical triplicates were run for detection of gene targets.Thermal cycling was performed using a MA6000 qPCR thermocycler (Sansure Biotech, Changsha, China).